Detection and Analysis of Somatic Mutations at a lacZ Reporter Locus in Higher Organisms

Application to Mus musculus and Drosophila melanogaster
  • Ana Maria Garcia
  • Rita A. Busuttil
  • Armando Rodriguez
  • Carlos Cabrera
  • Martha Lundell
  • Martijn E. T. Dollé
  • Jan Vijg
Part of the Methods in Molecular Biology™ book series (MIMB, volume 371)

Abstract

Methods to detect and analyze somatic mutations in higher organisms are critically important in view of their causal role in cancer, heritable diseases, and, possibly, aging. Here, we describe detailed protocols for the use of a mutational reporter system based on lacZ-containing plasmids integrated in the germline of Mus musculus and Drosophila melanogaster. Plasmids containing the bacterial lacZ gene integrated at one or more chromosomal sites can be excised, purified and recovered in suitable Escherichia coli hosts allowing the positive selection of mutant lacZ genes and their further molecular characterization. This system is capable of detecting a broad range of mutational events, varying from small mutations in the lacZ reporter gene to large genome rearrangements with one breakpoint in lacZ and the other breakpoint elsewhere in the genome.

Key Words

Somatic mutations genome rearrangements lacZ-plasmids Mus musculus Drosophila melanogaster 

References

  1. 1.
    Vijg, J. (2000) Somatic mutations and aging: a re-evaluation. Mutat. Res. 447, 117–135.PubMedGoogle Scholar
  2. 2.
    Vijg, J. and Dollé, M. E. (2002) Large genome rearrangements as a primary cause of aging. Mech. Ageing Dev. 123, 907–915.CrossRefPubMedGoogle Scholar
  3. 3.
    Fukuchi, K., Martin, G. M., and Monnat, R. J., Jr. (1989) Mutator phenotype of Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci. USA 86, 5893–5897.CrossRefPubMedGoogle Scholar
  4. 4.
    Gossen, J. A., de Leeuw, W. J., Tan, H., et al. (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc. Natl. Acad. Sci. USA 86, 7971–7975.CrossRefPubMedGoogle Scholar
  5. 5.
    Boerrigter, M. E., Dolle, M. E., Martus, H. J., Gossen, J. A., and Vijg, J. (1995) Plasmid-based transgenic mouse model for studying in vivo mutations. Nature 377, 657–659.CrossRefPubMedGoogle Scholar
  6. 6.
    Szilard, L. (1959) On the nature of the aging process. Proc. Natl. Acad. Sci. USA 45, 30–45.CrossRefPubMedGoogle Scholar
  7. 7.
    Dollé, M. E., Giese, H., Hopkins, L., Martus, H. J., Hausdorff, J. M., and Vijg, J. (1997) Rapid accumulation of genome rearrangements in liver but not in brain of old mice. Nat. Genet. 17, 431–434.CrossRefPubMedGoogle Scholar
  8. 8.
    Dollé, M. E., Snyder, W. Gossen, J. A., Lohman, P. H., and Vijg, J. (2000) Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine. Proc. Natl. Acad. Sci. USA 97, 8403–8408.CrossRefPubMedGoogle Scholar
  9. 9.
    Dollé, M. E. and Vijg, J. (2002) Genome dynamics in aging mice. Genome Res. 12, 1732–1738.CrossRefPubMedGoogle Scholar
  10. 10.
    Dollé, M. E., Martus, H. J., Gossen, J. A., Boerrigter, M. E., and Vijg, J. (1996) Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations. Mutagenesis 11, 111–118.CrossRefPubMedGoogle Scholar
  11. 11.
    Gossen, J. A., Martus, H. J., Wei, J. Y., and Vijg, J. (1995) Spontaneous and X-rayinduced deletion mutations in a LacZ plasmid-based transgenic mouse model. Mutat. Res. 331, 89–97.PubMedGoogle Scholar
  12. 12.
    Busuttil, R. A., Rubio, M., Dollé, M. E., Campisi, J., and Vijg, J. (2006) Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lac-plasmid reporter mice. DNA Repair 5(1), 52–60.CrossRefGoogle Scholar
  13. 13.
    Dollé, M. E., Snyder, W. van Orsouw, N. J., and Vijg, J. (1999) Background mutations and polymorphisms in lacZ-plasmid transgenic mice. Environ. Mol. Mutagen 34, 112–120.CrossRefPubMedGoogle Scholar
  14. 14.
    Dolle, M. E., Martus, H. J., Novak, M., van Orsouw, N. J., and Vijg, J. (1999) Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection. Mutagenesis 14, 287–293.CrossRefPubMedGoogle Scholar
  15. 15.
    Dollé, M. E., Busuttil, R. A., Garcia, A. M., et al. (2006) Increased genomic instability is not a prerequisite for shortened lifespan in DNA repair deficient mice. Mutat. Res. 596, 22–35.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Ana Maria Garcia
    • 1
  • Rita A. Busuttil
    • 2
  • Armando Rodriguez
    • 3
  • Carlos Cabrera
    • 3
  • Martha Lundell
    • 1
  • Martijn E. T. Dollé
    • 4
  • Jan Vijg
    • 2
  1. 1.University of Texas at San AntonioSan Antonio
  2. 2.Buck Institute for Age ResearchNovato
  3. 3.University of Texas Health Sciences CenterSan Antonio
  4. 4.National Institute of Public Health and EnvironmentBilthovenThe Netherlands

Personalised recommendations